Cellular cholesterol homeostasis is regulated at multiple cholesterol transfer steps and through a negative feedback loop that responds to elevations of membrane cholesterol in the endoplasmic reticulum (ER). Alterations in these sterol sensing and trafficking pathways in contribute to human inborn errors of metabolism and to acquired disease states. To elucidate mechanisms governing these critical cholesterol homeostatic pathways, we performed a functional genetic screen that led to isolation of cell lines with intracellular cholesterol trafficking defects that were enriched in mutants with disruption of long non-coding RNA (ncRNA) genes. These genes exhibit evolutionarily conserved exon/intron organization and core promoter regions, lack significant open reading-frames or nucleotide homology within exons, and contain short, highly conserved intronic regions that harbor small nucleolar RNA (snoRNA) species. This class of long ncRNAs appears principally to serve as host genes to facilitate expressing and processing of the orphan snoRNAs. We hypothesize that the processed snoRNAs modulate expression of genes involved in cholesterol homeostasis, possibly through control of splicing events or translation, and thus represent a previously unrecognized mode of regulation for cellular cholesterol homeostasis. The Specific Aims of this proposal are (1) To characterize ncRNA genes identified by our genetic screen that are critical for maintenance of cholesterol homeostasis, (2) To determine the role of the snoRNA host genes in regulation of intracellular cholesterol transfer and cellular cholesterol homeostasis, (3) To determine the molecular mechanism(s) through which the orphan snoRNA elements exert control over cholesterol regulatory pathways, and (4) To examine the physiological role of the orphan snoRNAs and gene pathways identified through the genetic screen by extending our cell-based studies to in vivo animal models. The proposed studies are innovative in that they explore a novel small RNA-dependent pathway not previously implicated in regulation of cellular cholesterol homeostasis. These studies are highly significant because elucidation of this RNA regulatory pathway has the potential to provide new molecular targets for manipulation of the cellular handling of cholesterol. This proposal is highly relevant to atherosclerosis, a common disease characterized by dysregulation of cholesterol homeostasis, as well as to rare, often fatal inborn errors of sterol metabolism.